Multi-drug resistant (MDR-) and extensively-drug resistant (XDR) Tuberculosis (TB) threaten to undo decades of progress, making the rapid detection of drug resistance crucial to TB control. Recent years have seen the development and deployment of nucleic acid technologies (NAT) that amplify and detect Mycobacterium tuberculosis (Mtb) nucleic acid (NA) directly from clinical samples. Although they require expensive and sophisticated instrumentation, NATs demonstrate sensitivities on par with culture, and yield results within hours rather than days or weeks. However, their cost prevents deployment to the peripheral labs where most patients seek diagnosis and treatment. In addition, multiple single nucleotide polymorphisms (SNP) must be simultaneously amplified, detected and discriminated from each other in order to identify resistant strains. To overcome these limitations, we designed a novel molecular reporter system, the SML-Generation Module (SGM), which can determine resistance to any drug and be formatted as a simple-to-execute kit. The SGM synthesizes a NA, the Surrogate Marker Locus (SML), as a surrogate marker for the phenotypic effects antimicrobials exert on susceptible organisms. This allows NATs to amplify and detect a single NA target to determine susceptibility of Mtb to a drug, dramatically simplifying NAT-based detection of drug resistance. The SGM is delivered to Mycobacteria by a recombinant mycobacteriophage, which we have shown can rapidly report the antibiotic susceptibility profile of cultured Mtb. In this application, we propose three Aims that will allow s to construct a second generation SGM (2SGM) reporter phage and assay capable of detecting </= 50cfu of Mtb per sample. We will then work with a subcontractor to create a research kit facilitating streamlined and reproducible testing of hundreds of clinical samples to establish the time to detection of the assay as well as its sensitivity and specificity in the determination of a complete front-line antibiotic susceptibility profile of Mtb directly from fresh clinical isolates.Aim I. Construction of a 2SGM reporter phage. Aim II. Development of a kit for standardized and reproducible front-line antibiotic susceptibility testing (AST) of clinical samples. Aim III. Clinial Evaluation of the SGM-phage kit for TB AST directly from patient samples. PUBLIC HEALTH RELEVANCE: Tuberculosis is the world's leading infectious disease killer and is becoming resistant to the drugs most effective in treating it. Successful treatment of this infection requires the creation of new technologies that can promptly and inexpensively assess which drugs will successfully kill the Tuberculosis bacteria inside a patient. We have invented a new technology with promise to accomplish this goal and are working to bring it to market.